Acetylcholine (ACh), a putative neurotransmitter in the central nervous system, is stored in multiple locations within the cholinergic nerve terminal, the cytosol and synaptic vesicles. The division of ACh into separate sites suggests that each site might have some physiological significance. Now it appears likely that the cytosol is the source of the ACh released from the nerve terminal under resting conditions, whereas the synaptic vesicle fraction is the source of the ACh released during nerve stimulation. Until recently, most cholinergic investigators believed that ACh was only formed in the cytosol by a soluble fraction of choline-0-acetyltransferase (ChAT) before being transported into synaptic vesicles for release. Recent evidence indicates that some of the nerve terminal ChAT is bound to synaptic vesicles and or plasma membranes; also, that this particulate ChAT fraction may play a critical role in the formation of ACh released in response to depolarization. The experimental designs described in this proposal will test the hypothesis that some of the membrane-bound ChAT in cholinergic terminals is anchored to synaptic vesicles by phosphatidylinositol; also, that the amount of ChAT associated in this way is regulated by an endogenous phospholipase C like enzyme, the activity of which is influenced by both depolarization and hyperpolarization of the cholinergic terminal. The significance of the proposed work will be to determine how ChAT is bound to synaptic vesicles, whether this particular ChAT fraction is important to the synthesis of ACh during nerve stimulation, and whether an endogenous system exists for the removal of ChAT from synaptic vesicle membranes. Answers to these questions will be helpful in clarifying the interrelationship between the different fractions of ChAT and their respective roles in the synthesis and release of ACh.